Cloning, expression and functional characterization of deoxyhypusine synthase from the pathogenic fungus Fusarium graminearum Schwabe (teleomorph Gibberella zeae), wheat (Triticum aestivum L.) and maize (Zea mays L.)

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Cloning, expression and functional characterization of

deoxyhypusine synthase from the pathogenic fungus Fusarium

graminearum Schwabe (teleomorph Gibberella zeae), wheat

(Triticum aestivum L.) and maize (Zea mays L.)

Dissertation

A thesis submitted for the degree of Dr. rer.nat. (rerum naturalium)

to the Biology Department,

the Faculty of Mathematics, Informatics and Natural Sciences,

University of Hamburg

By

Mayada Woriedh

Damascus, Syria

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When things get hard, let us not forget that-independent of race, colour, social situation, beliefs, or culture- everyone has experienced exactly the same. A lovely prayer written by the Egyptian Sufi master Dhu „I-Nun (d.ad 861) neatly sumps up the attitude one needs to adopt at such times:

O God, when I listen to the voices of the animals, to the sound of the trees, the murmur of the water, the singing of the birds, to the rushing of the wind or the rumble of the thunder, I see in them evidence of your unity; I feel that you are supreme power, supreme Knowledge, supreme wisdom, supreme justice.

O God, I also recognize you in the difficulties I am experiencing now. God, let your satisfaction be my satisfaction, and let me be your joy, the joy that a Father takes in his child. And let me remember You with calmness and determination, even when it is hard for me to say: I love You.

Paulo Coelho, Brazil Like the Flowing River, p.187

Be like the flowing river, Silent in the night. Be not afraid of the dark.

If there are stars in the sky, reflect them back. If there are clouds in the sky,

Remember, clouds, like the river, are water, So, gladly reflect them too,

In your own tranquil depths.

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I Summary

To date, not much is known about the signals necessary for the activation taking part in fungal pathogenicity of F. graminearum, which causes one of the most destructive crop diseases worldwide, and about the genes that are transcribed due to the activation of this pathway. Furthermore, plants respond to fungal pathogen ingress or abiotic stress by synthesizing defence proteins that facilitate stress healing, cell death or provide protection against further pathogen attack. The accumulation of these proteins is mainly due to rapid transcriptional activation of corresponding genes, though preliminary evidence implies that translational control may also have a role in the stress response. Pathogenic signal transduction mutants are a powerful tool for studying processes essential for pathogenicity. Further characterization of expression pattern comparisons between the wild-type strain and mutants will help to solve the open questions. This will lead to a better understanding of the infection mechanisms of this important plant pathogen and could, in the long term, enable to find new targets for F. graminearum-specific fungicides and to produce stress-resistant cultivars.

This study focuses on the function of the eukaryotic translation initiation factor 5A (eIF5A) and its hypusination through two enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydrolase (DOHH) in the phytopathogen Fusarium graminearum and in mechanical abiotic stress in wheat and maize and during infection with F. graminearum. Recent evidence suggests that eIF5A plays a role in protein synthesis and acts as a nucleo-cytoplasmic shuttling protein that facilitates mRNA translation through selective transport from the nucleus. eIF5A, DHS and DOHH have been identified and isolated in Fusarium graminearum as has DHS been identified and isolated in wheat Triticum aestivum and maize Zea mays, suggesting that each distinct gene may be involved in transport of different subsets of mRNA required for a specific physiological event.

The analysis in this study revealed that eIF5A, DHS and DOHH genes are important virulence factors as well essential for cell viability in Fusarium graminearum. Knockout of DHS and DOHH are lethal, while overexpression of DHS in Fusarium graminearum appears to be involved in the signal transduction pathways that result in aggressive virulent infection following high production of mycotoxins and reactive oxygen species, cell death, and severe penetrating wound of cell walls in host plants. In contrast, overexpression of DOHH in Fusarium graminearum appears to be involved in contrary transduction pathways that result in a weak infection following low production of mycotoxins and reactive oxygen species, low cell death, and lacking penetration of cell walls in host plants. Although overexpression of DHS

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increases the growth of Fusarium mutants under standard conditions and improves tolerance to high salt concentrations, it increases sensitivity to fungicide treatments. In constrast, overexpression of DOHH decreases the growth of Fusarium mutants, but increases tolerance to fungicide treatments. Moreover, this reduction does not restore correctly in DHS-DOHH double overexpression. DHS-DOHH overexpression appears to be balanced between DHS overexpression and DOHH overexpression, and adaptes in Fusarium mutants to survive under all stress conditions; oxidative stress, salt or fungicide. Also, it appears to result in an infection behavior more similar to infection patterns of Fusarim wild-type in host plants. Nevertheless, overexpression of these genes suppresses the growth of Fusarium mutants under temperature stress and produces lethal phenotypes.

Furthermore, the analysis in this study revealed that DHS in wheat and maize appears to be involved in the signal transduction pathways that result in cell death following virulent infection and systemically acquired resistance to F. graminearum, and systemically acquired resistance to salinity and drought stress. Transgenic wheat plants with reduced expression of DHS were developed using sense-linker-antisense T-DNA insertion by particle bombardment. In addition, transgenic maize plants with overexpression of DHS were developed too with double-stranded RNA-expressing constructs containing open reading frame of DHS introduced into maize by Agrobacterium mediated-transformation. Transgenic lines were isolated and propagated to be analyzed in a variety of assays. Analysis of the selectable marker expression revealed that most transgenes were transmitted faithfully. New cloning strategy was used to express the constructs by log on/off system of Cre-loxP with conditional promoters. The cloning strategy and frequencies of success of this large-scale project in wheat and maize, together with parameters for optimization at various steps, should serve as a useful framework for designing future RNAi or overexpression-based functional genomics projects in crop plants.

Finally, the capacity to effectively limit growth of various pathogens is important for the design of strategies to improve disease resistance and tolerance to abiotic conditions in crops. Development of resistant lines allows efficient crop production with reduced reliance on environmentally undesirable toxic agrichemicals. The strategy and the genes selected for testing in this study have not yet been described in wheat genome, maize genome or Fusarium genome.

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II List of Figures and Tables

Figure 1. Fusarium head blight disease cycle. ... 10

Figure 2. Wheat heads and maize ears with symptoms of Fusarium head blight.. ... 12

Figure 3. Evolution of hypusine biosynthesis in eIF5A. ... 17

Figure 4. Crystal structures of eIF5A. ... 19

Figure 5. Nucleotide and inferred amino acid sequences of the cDNA for senescence-induced wheat DHS.. ... 51

Figure 6. Sequence alignment of eIF5A genes.. ... 53

Figure 7. Amino acid alignment among putative DHS proteins. ... 54

Figure 8. Amino acid sequence conservation of DOHH in eukaryotes.. ... 55

Figure 9. Stress management. ... 57

Figure 10. Expression analysis of wheat DHS and Fusarium DHS, DOHH and eIF5A during infection of wheat spikes and non infected wheat plants... 59

Figure 11. Expression analysis of Fusarium eIF5A, DHS and DOHH compared with expression of Fusarium Tri4, Tri5, Tri6, Tri10 and Fgl1 in 1dpi-wheat spikes by semiquantitative RT-PCR. ... 60

Figure 12. Expression analysis of Fusarium DHS, DOHH and eIF5A genes in germinating conidia by semiquantitative RT-PCR. ... 60

Figure 13. Strategy for creating DOHH deletion construct.. ... 61

Figure 14. Scheme illustrating the generation of the DHS and DOHH overexpression constructs. ... 63

Figure 15. Southern blot analysis of the DHS, DOHH and double overexpressing mutants. .... 64

Figure 16. Confirmation by semiquantitative RT-PCR of overexpression of DHS, DOHH and double mutants.. ... 65

Figure 17. Morphological character and anastomosis events of Fusarium overexpressing mutants.. ... 67

Figure 18. Fusion of conidial anastomosis tubes and nuclei transfer. ... 68

Figure 19. Growth behavior of DHS, DOHH and double overexpressing mutants cultivated on CM at low temperatures.. ... 70

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Figure 20. Growth behavior of DHS, DOHH and double overexpressing mutants cultivated on

complete medium at different temperatures.. ... 72

Figure 21. Growth behavior of DHS, DOHH and double overexpressing mutants cultivated on YPG at different temperatures.. ... 74

Figure 22. Growth behavior of DHS, DOHH and double overexpressing mutants cultivated on poor medium (SNA) and SNA+1%NaCl.. ... 75

Figure 23. Growth behavior of DHS, DOHH and double overexpressing mutants cultivated on CM under salt stress. ... 77

Figure 24. Growth behavior of DHS, DOHH and double overexpressing mutants cultivated on CM with the fungicide Azoxystrobin.. ... 78

Figure 25. Cell death was measured spectrophotometrically by Evans blue staining. ... 80

Figure 26. Total protein measurement of DHS, DOHH, and double overexpressing mutants and Fusarium wilde-type strain 8/1 cultivated on rich media. ... 81

Figure 27. Pathogenicity tests of the overexpressing mutants on wheat spikes. ... 82

Figure 28. infection patterns of DHS, DOHH and double overexpressing mutants.. ... 83

Figure 29. DON measurement of 7 dpi-wheat spikelets with DHS, DOHH, and double overexpressing mutants and F.graminearum wild-type strain 8/1. ... 84

Figure 30. H2O2 measurement, ROS localization and response to oxidative stress. ... 86

Figure 31. Organization of the wheat ransformation vectors. ... 89

Figure 32. Location of the wheat DHS fragment used to construct the RNAi vector. ... 90

Figure 33. Cloning of the wheat DHS fragment selected for RNAi construct.. ... 91

Figure 34. Response of Florida to the tissue culture process.. ... 93

Figure 35. PCR screening of wheat transformants.. ... 94

Figure 36. Organization of the maize transformation vector... 96

Figure 37. Generation of transgenic maize plants. ... 98

Figure 38. Southern blot analysis of regenerated T0 maize plants.. ... 99

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III List of Abbreviations

% Percentage A Adenine aa Amino acid Amp Ampicillin ATP Adenosintriphosphate

bar phosphinothricin acetyl transferase

BLAST Basic Local Alignment Search Tool

bp Base pairs

°C Degree Celsius

C Cytosine

CaMV Cauliflower mosaic virus

cDNA complementary Deoxyribonucleic Acid

CIMMYT International Maize and Wheat Improvement Center

CM Complete medium

CNI Guanylhydrazone CNI-1493

Cre Cre recombinase

CTAB Cetyl trimethyl ammonium bromide

cv. Cultivated variety; cultivar

2,4-D 2,4-Dichlorophenoxy acetic acid

DHS Deoxyhypusine synthase

DIG Digoxygenin

DMSO Dimethylsulfoxid

DNA Deoxyribonucleic acid

dNTPs Deoxynucleotide triphosphate (s)

DOHH Deoxyhypusine hydroxylase

DON Deoxynivalenol

dpi days post inoculation

DsRed Discosoma sp. red fluorescent protein

dsRNA double stranded RNA

dUTP Deoxyuracil triphosphate

E. coli Escherichia coli

EC Enzyme Commission

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eIF5A Eukaryotic initiation factor 5A

et al. et alii = and others

Fgl Fusarium graminearum lipase

FHB Fusarium Head Blight

g Gram; the metric unit of mass

G Guanine

GC- guanosine-cytosine content

GC7 N1-guanyl-1,7-Diaminoheptan

gDNA genomic DNA

GFP Green fluorescent protein

Gpmk1 Gibberella pathogenicity MAP kinase 1

GUS Glucuronidase

hph Hygromycin B phosphotransferase

HSP Heat shock promoter

HSS Homospermidin synthase

IR Inverted repeat region

kb kilo bases (= 1000 bp)

kDa kilo Dalton (= 1000 Da)

L Liter

LB Luria-Bertani medium

M Molar (mol/L)

MAP Mitogen Activated Protein

MAPK Mitogen Activated Protein Kinase

Min. Minute

MIPS Munich Information center for Protein Sequences

miRNA microRNA

ml milliliter

mM millimolar

MOPS 3-(N-Morpholino) propane sulfonic acid

mRNA messenger RNA

NAD+ Nicotinamide adenine dinucleotide

NCBI National Center for Biotechnology Information

NIV Nivalenol

nptII neomycin phosphotransferase

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ORF Open Reading Frame

PCR Polymerase chain reaction

PEG Polyethylene glycol

pH Potentia Hydrogenii

PTGS post-transcriptional gene silencing

RISC RNA-induced silencing complex

RNA Ribonucleic acid

RNAi RNA interference

ROS Reactive oxygen species

rpm round per minute

rRNA ribosomal RNA

RT room temperature

RT-PCR reverse transcription - polymerase chain reaction

SDS Sodium dodecyl sulphate

siRNA small interfering RNA

SNA Synthetic Nutrient-poor mineral Agar

sRNA short RNA

ssRNA single stranded RNA

T Thymine

TBE TRIS-Borate-EDTA

Tm Annealing Temperature

Tri Trichothecene synthase gene

Tris Tris-(hydroxymethyl) aminomethane

tRNA transfer RNA

UniProtKB Protein knowledge base

UTR Untranslated region

UV Ultra violet

v Volume

v/v volume per volume

w/v Weight per volume

WT Wild-type

YPG Yeast-extract Peptone Glucose

Units of measurements were used according to the International System of Units SI (Système International d‟Unités). Chemical formulas and molecules are named after IUPAC (International Union of Pure and Applied Chemistry).

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1.0 Introduction

1.1 Fusarium head blight

Fusarium head blight (FHB), also known as scab or tombstone, is a disease of wheat, barley, oats and other small cereal grains and corn (Leslie and Summerell, 2006; Walter et al, 2010). It is caused by several species of Fusarium; however, Fusarium graminearum is the primary species involved. This disease reduces yield and grade and may also contaminate the grain with fungal toxins (mycotoxins). Fusarium head blight also negatively affects grain

quality, often resulting in lower test weights and mycotoxin contamination led a majority of the

crop rejected by the industry (Jansen et al, 2005; Leslie and Summerell, 2006). FHB is favoured by humid conditions during flowering and early stages of kernel development (De Wolf, 2003; Jansen et al, 2005). Fusarium head blight (FHB) or scab of barley and wheat is considered the worst plant disease in the U.S. since the stem rust epidemics of the 1950s (Windels, 2000). The economic losses caused by scab epidemics during 1993–1998 for wheat and barley farmers in the Midwest are estimated at $3 billion. The epidemics led to barley yield decreases from 75.4 to 46.5 bushels per acre in North Dakota and from 76.2 to 60.2 bushels per acre in Minnesota. Wheat yields dropped in the two states by 48% and 39%, respectively (Jansen et al, 2005; McMullen et al, 2008).

In most areas, FHB is caused by a complex of various Fusarium species differing in important biological and ecological characteristics, e.g. virulence on cereals, host range, mycotoxin production, optimum growth conditions, survival on crop debris and in the soil

(Ghahderijani, 2008). When cereal plants are infected with these fungi, there is a risk that grain may become contaminated with Fusarium mycotoxins, which may subsequently be transferred

to compound feeds (Ghahderijani, 2008) The distribution and predominance of FHB pathogens

differ significantly among climatic conditions, geographical zones, countries, and years (Xu and Berrie, 2005; Giraud et al, 2010)

In Germany, five Fusarium species have been reported to predominate in the FHB complex

(Schollenberger et al, 2006; Ghahderijani, 2008): Fusarium graminearum Schwabe (teleomorph = Gibberella zeae (Schwein.) Petch), F. culmorum (Smith) Sacc, F. avenaceum

(Corda ex Fr.,) (teleomorph = G. avenaceum Cook), F. poae (Peck) Wollenw, and

F. tricinctum (Corda) Sacc) (teleomorph = G. tricincta El-Gholl, McRitchie, Schoult. &

Ridings). F. graminearum is the primary species involved in FHB and the predominant species

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Summerell, 2006; Ghahderijani, 2008; Becher et al, 2010). Environmental factors such as water activity, temperature, relative humidity, irradiation and pH value may influence the spectrum of different Fusarium species on wheat ears and in different areas and also their

interactions (Cumagun, 2004).

1.1.1 The fungus Fusarium graminearum: Taxonomy, Pathology and Ecology

Gibberella zeae (Schwein.) Petch, (anamorph: Fusarium graminearum Schwabe) is a filamentous ascomycete that infects diverse plant species and those of economic importance

include maize and small grains such as wheat, barley, rye, triticale, rice, but is also known from other annual and perennial plants (Cumagun, 2004; Cumagun and Miedaner, 2004). Fusarium graminearum can be a pathogen on the model plant species Arabidopsis thaliana, which may enable more rapid studies of host-pathogen interactions for this fungus (Leslie and Summerell, 2006). In wheat, G. zeae causes seedling blight, crown rot, root rot, and head blight (Cumagun, 2004). In barley and oats, G. zeae causes Fusarium mould and head blight (De Wolf, 2003), Also, it causes red cob in maize. Maize lines vary widely in their sensitivity to F. graminearum (Leslie and Summerell, 2006).

Life and disease cycle. F. graminearum is a haploid homothallic ascomycete. The fruiting

bodies called perithecia develop on the mycelium and give rise to ascospores, which land on susceptible parts of the host plant and germinate (Figure 1). The fungus causes head blight on wheat, barley, and other grass species as well as ear rot on corn. The primary inoculum are the ascospores, sexual spores which are produced in the perithecia (Beyer and Verreet, 2005; Gilbert et al, 2008). Outcrossing enables the fungus to have a high genotypic diversity, which allows natural populations to adapt faster to selective pressures, such as cultivar resistance or fungicides (Jenczmionka et al, 2003). Spores are forcibly discharged and can germinate within six hours upon landing on the plant surface. The scab disease is monocyclic; after one cycle of infection with ascospores, the fungus produces macroconidia by asexual reproduction (Beyer et al, 2004). These structures overwinter in the soil or in plant debris on the field and give rise to the mycelium in the next season. The colonization of the other crops and grasses is important because the fungus survives in the crop residues that remain on the soil surface. The fungus reproduces in the crop residues and is moved by wind or rain to the developing wheat, barley or maize. Wheat is most susceptible during the flowering growth stages (Jansen et al, 2005), but some infection can still occur during kernel development. Temperatures between 19 and 30°C and extended periods of moisture in the form of rain or dew favor reproduction of the fungus on crop residues and also promote infection and disease development. Disease development is

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favoured when cool wet weather occurs during short periods (10–20 days) from anthesis through the soft stage of kernel development in wheat and barley and during the first 21 days after silking in maize (De Wolf et al, 2003; Jenczmionka et al, 2003; Leslie and Summerell, 2006).

Figure 1. Fusarium head blight disease cycle. (Life cycle composition by Shaobin Zhong; NDSU Department of

Plant Pathology, North Dakota State University, USA, 2008).

Formation of mycotoxins. F. graminearum species are responsible throughout the world for

the formation of mycotoxins in infected plants and in plant products (Giraud et al, 2010; Lehoczki-Krsjak et al, 2010; Schmidt-Heydt et al, 2010). When cereal plants are infected with these fungi, there is a risk that grain may become contaminated with Fusarium mycotoxins, which may subsequently be transferred to compound food and feed (Bernhoft et al, 2010;

Moretti et al, 2010). Isolates of F. graminearum may produce three important mycotoxins,

zearalenone, nivalenol and deoxynivalenol as well as aurofusarin, culmorins, fusarin C, fusarochromanone, and steroids (Leslie and Summerell, 2006). The molecular genetics of trichothecene production is generally well understood and is regulated by the Tri6 and Tri5 gene product (Matsumoto et al, 1999; Brown et al, 2004). Production of deoxynivalenol or nivalenol is usually determined by the functioning of a single gene, Tri13, Tri7 (Lee et al, 2001b; Lee et al, 2002; Kim et al, 2003)(1056, 1209, 1210), while Tri14 is required for high virulence and DON production but not for DON synthesis in vitro (Dyer et al, 2005).

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F. graminearum produces trichothecenetype A toxins (HT-2 toxin, T-2 toxin) and type B toxins (deoxynivalenol, 3-acetylnivalenol, 15-acetyldeoxynivalenol, nivalenol, fusarenone-X, calonectrin) and zearalenon (Cumagun, 2004; Ghahderijani, 2008). The difference between of type A and B is the absence or presence of a keto group at C-8 of the trichothecene skeleton,

respectively (Cumagun, 2004; Ghahderijani, 2008). Type B toxins could be subdivided into

two major chemotypes: (1) nivalenol chemotype which produces nivalenol (NIV) and fusarenone-X and (2) deoxynivalenol chemotype which produces deoxynivalenol (DON,

vomitoxin) and acetyldeoxynivalenol (ADON) (Cumagun, 2004; Ghahderijani, 2008). As a member of the sesquiterpenoid family of natural products, trichothecene toxins such as DON

are potent eukaryotic protein inhibitors (Wache et al, 2009) causing decreased food consumption and lower weight gain in animals followed by diarrhea, vomiting, reproductive and haematological problems (Ansari et al, 2009). Human ingestion of contaminated grains is associated with alimentary toxic aleukia, nausea, depression of the immune system, skin necrosis and hemorrhage of lungs and gastrointestinal tract (Donmez-Altuntas et al, 2007; Celik et al, 2009; Schwerdt et al, 2009). Trichothecenes are also phytotoxins causing chlorosis, necrosis, and wilting (Cumagun and Miedaner, 2004). For this reason, these compounds might contribute to the pathogenicity and/or aggressiveness of the pathogen. DON producers of F. graminearum are considered more aggressive on wheat and rye than NIV producers (Cumagun and Miedaner, 2004).

The fungal toxin zearalenone has estrogenic properties and produces many reproductive disorders in animals. Swine are the most sensitive to the toxin, but cattle and sheep may also be affected. Zearalenone concentrations of 1–5 ppm can result in negative effects in animals and humans. Industries concerned about mycotoxins test grain prior to feeding to animals (De Wolf et al, 2003; Urraca et al, 2005). When the environmental conditions favor the fungal infection and later on development, the concentration of ZEA can dramatically increase either in field or storage conditions. Zearalenone is not degraded in common food and feed processing procedures, as it has been shown its presence in grain products like bread, locally brewed beers and processed feeds (Malekinejad et al, 2007; Sabater-Vilar et al, 2007)

1.1.2 Syptoms and infection process of F. graminearum on cereal crops

FHB is recognized in the field by the premature bleaching of infected spikelets and the production of orange, spore-bearing structures called sporodochia at the base of the glumes (Jansen et al, 2005; Leslie and Summerell, 2006). During wet weather, there may be whitish, occasionally pinkish, fluffy fungal growth on infected heads in the field. Diseased spikelets can

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contain visibly affected kernels (Leslie and Summerell, 2006). The grading term given to visibly affected wheat seeds is fusarium damaged kernels (FDK), whereas in barley, it is called Fusarium mould (De Wolf, 2003; Xu and Berrie, 2005). FDK in wheat are shrunken and typically chalky white in appearance, hence the name “scab” (Cumagun, 2004), while fusarium mould on barley is an orange or black encrustation of the seed surface (De Wolf, 2003; Jansen et al, 2005). Much of the severely infected wheat is poorly filled and may be blown out the back of the combine due to its low kernel weight. Grain infected wheat after the flowering stage may be heavy enough to be harvested along with healthy grain, although mostFDK will result from infections that occur during flowering. The fungus may eventually kill the developing seed at about the soft dough stage. Symptoms of Fusarium mould in barley or oats are usually sparse, making it difficult to tell if grain has been infected (De Wolf, 2003; Lewandowski et al, 2006). In maize, F. graminearum infects maize ear through silk channels and is most efficient when the silks are newly emergent causing ear rot (Figure 2) (Velluti et al, 2000; Hartmann et al, 2008).

Figure 2. Wheat heads and maize ears with symptoms of Fusarium head blight. From left to right: diseased

spikelets become bleached or tan in appearance and may have signs or fungal reproduction; orange structure at the base of the diseased spikelet; white mold characteristic of Fusarium ear rot. Wheat photos (De Wolf, 2003). Maize photo (Department of Plant Pathology, University of Illinois, USA, 2008).

F. graminearum infects wheat spikes from anthesis through the soft dough stage of kernel development. The fungus enters the plant mostly through the flowers. However, the infection process is complex and the complete course of colonization of the host has not been described. Germ tubes seem not to be able to penetrate the hard, waxy surface of the lemma and palea which protect the flower. The fungus enters the plant through natural openings like stomates and needs soft tissue like the flowers, anthers and embryo to infect the plant (Bushnell and Leonard, 2003). From the infected floret, the fungus can grow through the rachis and cause severe damage in a short period of time under favorable conditions. Upon germination of the

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spores on the anthers and the surface of the developing kernel, hyphae penetrate the epicarp and spread through the seed coat. Successively, the different layers of the seed coat and finally the endosperm are colonized and killed (Jansen et al, 2005). The first symptoms include a tan or brown discoloration at the base of a floret within the spikelets of the head. As the infection progresses, the diseased spikelets become light tan or bleached in appearance (Figure 2). The infection may be limited to one spikelet, but if the fungus invades the rachis the entire head will develop symptoms of the disease. The base of the infected spikelets and portions of the rachis often develop a dark brown color. When weather conditions have been favorable for pathogen reproduction, the fungus may produce small orange clusters of spores or black reproductive structures called perithecia on the surface of the glumes (De Wolf, 2003; Cumagun and Miedaner, 2004). If the heads invade extensively at early stages, kernels will fail to develop entirely.

1.1.3 Virulence factors of F. graminearum and plant interactions

Trichothecene production is an important character for virulence and associated with increased disease severity and for spread of the fungus within an infected wheat head (Maier et al, 2006). F. graminearum mutants with a disrupted Tri5 gene were shown to be unable to produce deoxynivalenol and to be drastically reduced in virulence (Jansen et al, 2005; Maier et al, 2006). In the absence of trichothecenes, the fungus is blocked by the development of heavy cell wall thickenings in the rachis node, a defense inhibited by the mycotoxin (Jansen et al, 2005). In wheat, the toxins were detected in association with cytosolic ribosomes, chloroplasts, plasmalemma, cell walls, and vacuoles. The number of enzymes examined from F. graminearum, other than those associated with toxin biosynthesis, is not large and includes xylanases (Belien et al, 2005). Toxins were transported apically in xylem and phloem of the rachis to distal uninfected florets (Kang et al, 2001). With the aid of antibodies reacting with cellulose, xylans, and pectin, it was shown that the cell walls of infected host cells were degraded. This finding provides evidence for the release of enzymes from the pathogen for digestion of cell walls at early stages of infection (Jansen et al, 2005). Therefore, these fungi rely on other penetration mechanisms, e.g. the enzymatic digestion of the plant cell wall. This makes necessary the secretion of hydrolyzing enzymes, like cutinases, cellulases, amylases and pectinases (Jenczmionka et al, 2003). The MAP kinase encoded by Gpmk1 regulates the expression of secreted cell-wall degrading enzymes required for pathogenicity (Leslie and Summerell, 2006) as well as the secreted lipase Fgl1, a major virulence factor of Fusarium graminearum (Jenczmionka et al, 2003). MAP kinases belong to the family of serine/threonine protein kinases. They are activated by a MAPKKK-MAPKK-MAP kinase cascade. This

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cascade is conserved in eukaryotic organisms and is involved both in the transduction of a variety of extracellular signals and in the regulation of growth and differentiation process

(Jenczmionka et al, 2003). Fungal MAP kinases have regulating functions, ranging across conidiation and conidia germination, appressorium and penetration peg formation, invasive hyphal growth and response to hyperosmotic stress and cell turgor (Jansen et al, 2005; Leslie and Summerell, 2006). Furthermore, MAP kinases have also been reported to play an important role during the regulation of mating (Muller et al, 1999).

F. graminearum can utilize a broad range of compounds as the sole nitrogen source including nitrate, ammonium, urea, and most purines and amino acids that do not contain sulfur (Leslie and Summerell, 2006). It has a fairly typical fungal cell wall composed of chitin and cross-linked galactose, mannose, arabinose and glucuronic acid (Leslie and Summerell, 2006), while it is also sensitive to the plant defense chemical apigeninidin (Leslie and Raju, 1985). Increased ferulic acid content in maize kernels is correlated with resistance to the ear rot induced by F. graminearum (Leslie and Summerell, 2006). Labeling of wheat 1,3-ß-glucanase and chitinase with antibodies against the tobacco enzymes indicated that these enzymes were induced during infection in wheat and barley, and considered to be partially resistant to Fusarium (Anand et al, 2003; Kong et al, 2005; Voigt et al, 2006; Shin et al, 2008). Also, it was suggested that FHB resistance in wheat involves defense pathways regulated by jasmonic acid and ethylene signaling, while salicylic acid–regulated systematic acquired resistance is insignificant in this process (Guangle and Yang, 2008).

In general, the first step in plant infection is perception of the host by the fungal pathogen. This is mediated by fungal receptors that recognize physical and/or chemical signals from the plant, leading to responses needed for successful infection, e.g. the development of penetration structures and/ or the secretion of cell wall-hydrolyzing enzymes. Plant perception and expression of proteins necessary for infection are linked by signal transduction pathways, which, then, are likely to be a crucial factor in disease establishment. (Jansen et al, 2005). After entry of a pathogen, the plant responds to infection in differnt ways. (1) If the plant is resistant, F. graminearum will not be able to replicate, thrive and cause disease in the plant (Hermann and Day, 2001). (2) F. graminearum may be able to cause a systemic infection, where the pathogen replicates and spreads from the initially infected cells to other cells of the plant showing physical symptoms and interferes with growth and development of the plants, leading to a widespread chlorosis, necrosis and bleaching (Hermann and Day, 2001). (3) An interaction can also occur between plants and pathogens when the plant actively combats the fungal pathogen through activation of specific disease resistance signaling by a hypersensitive

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response and avirulence factors. In this process, the plant recognizes that it is being invaded by a foreign organism and immediately initiates a response by which the growth of the pathogen is suppressed (Hermann and Day, 2001; Sella et al, 2004). The cells surrounding the infection site immediately undergo programmed cell death causing necrotic lesions to localize the invading pathogen in the dead tissue and to prevent movement from cell to cell with association of the oxidative burst. The production of reactive oxygen species (ROS) is a downstream component of the hypersensitive response of the plant reaction to the oxidative burst (Morel and Dangl, 1997; Hermann and Day, 2001). Also, host cell death can occur in both susceptible (compatible) and resistant (incompatible) plant-pathogen interactions. Several studies indicate that cell death during the hypersensitive response involves activation of a plant-encoded pathway for cell death (Morel and Dangl, 1997). The cellular characteristics of the death process strongly implicate specific signals and autonomous cellular biochemical processes that execute individual cells (Gatsukovich, 2004). Apoptosis is genetically controlled cellular suicide essential for development, maintenance of cellular homeostasis, and defense against environmental insults including pathogen attack (Morel and Dangl, 1997; Gatsukovich, 2004). Some features of programmed cell death have been observed in both susceptible and resistant reactions during plant-pathogen interactions, suggesting negative regulators of apoptosis exhibited heritable resistance to several necrotrophic fungal pathogens (Hermann and Day, 2001; Gatsukovich, 2004) that overlap biochemical pathways operative in these two contrasting outcomes. Suppression of plant cell death or senescence may improve resistance to abiotic stress and necrotrophic pathogens such as F. graminearum.

Hence, hyphae of germinating fungal spores use different paths of infection. Effective resistance to Fusarium head blight requires expression of genes that combat these different pathways of infection. More detailed studies are required to evaluate the possibilities for targeting metabolites or the elimination of mycotoxins or for using defense genes known to be effective against various necrotrophic or biotrophic pathogens. Recently, several studies have shown that infection by F. graminearum induces transcript accumulation of several classes of biotic and abiotic stress-related genes in both partially resistant and susceptible cultivars (Kong et al, 2005). The expression of biotic and abiotic stress-related genes may result in a reduction of FHB severity in wheat, but the relationship and mode of interaction between FHB resistance and F. graminearum has not been clearly established. Identifying host genes differentially expressed in response to the pathogen may help illustrate cellular processes activated or repressed during the early phase of host–pathogen interactions that ultimately determine the extent of fungal colonization. At present, no highly resistant cultivars are available and not much is known about genes involved in the process of infection and plant interactions.

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1.2 Hypusine biosynthesis pathway

The only hypusine-containing protein identified so far is the eukaryotic initiation factor 5A (eIF5A) (Park et al, 2010). Hypusine is formed in a novel posttranslational modification that involves two enzymes, deoxyhypusine synthase (DHS), and deoxyhypusine hydroxylase (DOHH). DHS (EC 2.5.1.46) catalyzes the cleavage of the polyamine spermidine and transfer of its 4-aminobutyl moiety to the ε-amino group of one specific lysine residue of the eIF5A precursor to form a deoxyhypusine intermediate. In the next step, DOHH (EC 1.14.99.29) converts the deoxyhypusine-containing intermediate to the hypusine-containing mature eIF5A. Forming of hypusine results in activation of eIF5A (Figure 3) (Park, 2006; Wolff et al, 2007).

Polyamines, i.e., putrescine, spermidine, and spermine, are ubiquitous in living cells and are essential for cell growth. These polyamines exist as protonated polycations at physiological pH in cells and interact with nucleic acids, DNA and RNA, acidic proteins, and phospholipids (Huang et al, 2007; Park et al, 2010). Polyamines regulate a vast array of cellular activities at the level of replication, transcription, translation, posttranslational modification, protein activation, membrane stability, and ion channelling (Park, 2006; Park et al, 2010). The role of polyamines in translational regulation may be most important, because a majority of polyamines are bound to RNA (Igarashi and Kashiwagi, 2009) and in that the polyamine spermidine is required for activation of the eukaryotic initiation factor 5A (eIF5A). Thus, polyamines regulate cell growth, proliferation, differentiation, transformation, and apoptosis and their cellular levels are tightly regulated at the level of biosynthesis, metabolism, and transport (Park et al, 2010). In eukaryotic organisms, the polyamine spermidine has an independent and specific function as the source of the 4-aminobutyl portion of hypusine [Nε-(4-amino-2-hydroxybutyl)-lysine] in the essential cellular protein eIF5A (Park, 2006; Kang et al, 2007; Wolff et al, 2007). Since the structural requirement for spermidine in the synthesis of hypusine is quite strict, this function can only be fulfilled by spermidine or by few close structural analogs. Thus, hypusine synthesis defines an absolute requirement for the polyamine spermidine in eukaryotes (Byers et al, 1994; Chattopadhyay et al, 2003; Park et al, 2010).

Hypusine was first discovered in bovine brain (Shiba et al, 1971). An 18,000-dalton protein was found to contain hypusine (Park et al, 1981). The 18,000-dalton protein was found to be eIF-4D (Cooper et al, 1983). eIF-4D was renamed as eIF-5A (1987). eIF-5A was nemaed as eIF5A (1996). eIF5A, DHS and DOHH are highly conserved proteins from archaebacteria to mammals, suggesting a vital function of eIF5A and the deoxyhypusine/hypusine modification. Deoxyhypusine and hypusine occur in archaea and eukaryotes, but not in eubacteria (Klier and

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Lottspeich, 1992; Park, 2006; Park et al, 2010). Yet, eubacteria have an elongation factor P (Figure 3), which is a distant ortholog of eIF5A. EF-P enhances elongation by stimulating the peptidyl transferase activity of the ribosome and is an essential protein in bacteria. In plant, hypusine has been described so far in Arapidopsis, tomato, rice and tobacco (Ober and Hartmann, 1999; Wang et al, 2001; Wang et al, 2003; Wang et al, 2005b; Duguay et al, 2007). In fungi, hypusine has been described in the filamentous fungi Neurospora crassa and in the slime mould Dictyostelium discoideum (Mackintosh and Walters, 2003).81) n 18,000-dound

to

Figure 3. Evolution of hypusine biosynthesis in eIF5A. Upper panel, the polyamine spermidine, which is

synthesized from putrescine, is the source of the aminobutyl moiety of hypusine, as indicated by shading. Hypusine synthesis occurs at one specific lysine residue of the eIF5A precursor protein, eIF5 (Lys), by two enzymatic steps, involving deoxyhypusine synthase (DHS) and deoxyhypusin hydroxylase (DOHH). Deoxyhypusine synthase (DHS) reaction is reversible, whereas the deoxyhypusine hydroxylase (DOHH) reaction is not. Lower panel, Evolution of eIF5A and its hypusine modification pathway. eIF5A orthologs are found in eubacteria and archaea and are essential genes in each organism. The DHS gene exists in archaea, and in all eukaryotes, but not in eubacteria (elongation factor). DOHH gene is found only in eukaryotes. E indicates essential gene, and NE indicates non-essential gene. The Figure is illustrated after (Park et al, 2010).

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1.2.1 Eukaryotic initiation factor 5A (eIF5A)

To elucidate the biological function of eIF5A, many attempts have been made, but its exact function remains mysterious. Earlier molecular genetics and biochemical studies described eIF5A as a translation initiation factor due to its ability to stimulate the synthesis of

methionyl-puromycin in vitro (Park et al, 1993a, 1993b). Later, depletion of eIF5A in yeast caused only a small (30%) reduction in the protein synthesis rate (Kang and Hershey, 1994). These results

have been used to argue against eIF5A as a translation initiation factor for general protein

synthesis. More recent evidence showed that hypusine-containing eIF5A is a nucleocytoplasmatic shuttle protein of a subset of mRNAs related to the G1/S cell cycle

transition and unspliced viral mRNAs, and promoting nuclear export of specific mRNA facilitates protein synthesis and facilitates their translation. It has also been shown that eIF5A may be involved in mRNA turnover, acting downstream of decapping (Ruhl et al, 1993; Hanauske-Abel et al, 1995; Bevec et al, 1996; Jin et al, 2003; Luchessi et al, 2006; Schrader et al, 2006; Njuguna, 2009). Recently, hypusinated eIF5A contributes to the life cycle of human immunodeficiency virus by interacting with the retroviral REV protein, thereby participating in the nuclear export of unspliced and incompletely-spliced viral mRNA. Hence, human eIF5A may form part of a specific nuclear export pathway that is exploited by the virus for propagation (Liu et al, 1997; Hart et al, 2002; Hauber et al, 2005). In addition, immunocytochemical analyses showed differences in the distribution of non-hypusinated eIF5A precursor and the hypusine-containing mature eIF5A. While the precursor is found in both cytoplasm and nucleus, the hypusinated eIF5A is primarily localized in cytoplasm (Lee et al, 2009). These findings provide strong evidence that the hypusine modification of eIF5A dictates its localization in the cytoplasmic compartment where it is required for protein synthesis.

The X-ray of the crystal structure of the eIF5A (Park, 2006) reveals that this protein consists of two well-defined domains: the N-terminal domain, which contains the hypusine modification site in an exposed loop, and the C-terminal domain, which is similar to the oligonucleotide-binding domain found in several RNA-binding proteins (Figure 4). RNA binding depends on both the presence of the hypusine residue in the eIF5A protein and conserved core motifs of the target RNA (Park, 2006; Parreiras et al, 2007).

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Figure 4. Crystal structures of eIF5A. X-ray structure of eIF5A precursor containing two domains: N-terminal

domain (I) and C-terminal domain (II), the location of the lysine residue (in domain I) that undergoes modification to deoxyhypusine or hypusine is indicated (Park, 2006).

The most convincing evidence for the essential nature of eIF5A and its deoxyhypusine/hypusine modification was obtained from gene mutation, gene disruption or knock down studies in the yeast S. cerevisiae and higher eukaryotes (Schnier et al, 1991; Sasaki et al, 1996; Frigieri et al, 2007; Frigieri et al, 2008), and proved the essentiality of these genes for cell viability, cell growth, differentiation and the efficiency for proliferation (Park et al, 1993a, 1993b; Dias et al, 2008). Further studies demonstrated that yeast eIF5A is involved, too, in vesicular trafficking, cell wall integrity and reveals connections to poly(A)-binding protein, to protein kinase C signaling and to the secretory pathway (Valentini et al, 2002; Zanelli and Valentini, 2007; Frigieri et al, 2008). Currently, this gene also evidenced to promote translation elongation and ribosomal transit times and is required for the first peptide bond of protein synthesis (Cano et al, 2008; Frigieri et al, 2008; Lee et al, 2009; Saini et al, 2009).

There are two known eIF5A isoforms in humans, three in Arabidopsis, and four in lettuce (Thompson et al, 2003; Wang et al, 2003). Little is known about eIF5A in plants, although it has been cloned from alfalfa, tobacco, canola, tomato and Arabidopsis (Wang et al, 2001; Gatsukovich, 2004; Thompson et al, 2004; Lebska et al, 2009). It was observed that the transcript levels of eIF5A increased during natural and stress-induced senescence in tomato (Wang et al, 2001). In Arabidopsis, recent evidence indicates that eIF-A-1 plays a pivotal role in cell proliferation and senescence (Wang et al, 2003; Thompson et al, 2004). Separate isoforms of eIF5A appear to facilitate the translation of mRNAs required for cell division and cell death. This raises the possibility that eIF5A isoforms are elements of a biological switch that is in one position in dividing cells and in another position in dying cells. Changes in the position of this putative switch in response to physiological and environmental cues are likely to have a significant impact on plant growth and development (Thompson et al, 2004). Futhoremore, eIF5A-2 is involved in pathogen-induced cell death and development of disease symptoms in Arabidopsis during infection with Pseudomonas syringae pv. tomato (Hopkins et

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al, 2008). Moreover, constitutive downregulation of eIF5A-1 resulted in the major suppression of xylem formation, and the antisense transgenic plants were also stunted. These data collectively indicate that modulation of eIF5A-1 expression alters xylem abundance in Arabidopsis thaliana (Liu et al, 2008).

The discovery of various isoforms of eIF5A has given rise to the distinct possibility that

each isoform is responsible for transporting a unique subset of mRNA species required for a specific physiological event, such as cell division, cell death or pathogen attack. In effect,

each eIF5A isoform is thought to act as a shuttle protein that initiates the translation of a developmentally required suite of genes (Parreiras et al, 2007).

1.2.2 Deoxyhypusine synthase (DHS)

Deoxyhypusine synthase catalyzes a complex sequence of reactions, involving two substrates, spermidine and eIF5A (Lys), and a cofactor, NAD, to convert one specific lysine residue of the eIF5A precursor to a deoxyhypusine residue. This enzyme exhibits an absolute specificity toward its protein substrate, eIF5A(Lys) (Park, 2006). DHS, like eIF5A, is well conserved through evolution and present in all eukaryotes, in certain archaea, and even several cyanobacteria (Wolff et al, 2007). Like the plant deoxyhypusine synthase from tobacco and Senecio vernalis, human DHS can accommodate putrescine as an alternative butylamine acceptor instead of eIF5A(Lys) resulting in the formation of homospermidine from spermidine (Park, 2006). However, it was demonstrated that deoxyhypusine synthesis is the preferred pathway of the DHS reaction (Park, 2006). Furthermore, all the DHS-catalyzed reactions are reversible and DHS can facilitate interconversion of spermidine, eIF5A (intermediate) and homospermidine by way of a common enzyme-imine intermediate (Park et al, 2010). Thus, the aminobutyl moiety of the enzyme-imine intermediate can be transferred to any one of the three acceptors, eIF5A (Lys), putrescine or 1,3-diaminopropane leading to the synthesis of deoxyhypusine, homospermidine or spermidine, respectively (Park, 2006) (Figure 3).

DHS has been found to be involved in cell proliferation via its catalytic role in the hypusination pathway of eIF5A and play a role in another independent cellular function as well (Park, 2006). There is only a single DHS gene in yeast and most eukaryotes. A haploid S. cerevisiae strain with disruption of the DHS gene was not viable (Wöhl et al, 1993; Sasaki et al, 1996; Park et al, 1998). Growth was arrested in G1 upon treatment with GC7, a potent inhibitor of DHS (Mackintosh and Walters, 2003), indicating that DHS is also essential. The inhibition of deoxyhypusine synthase by inhibitors results in suppressed growth of actively

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dividing mammalian cells and demonstrates an antiproliferative potential (Park et al, 1994; Wolff et al, 2007). Screening assays of deoxyhypusine synthase inhibitors identified the guanylhydrazone CNI-1493, a novel inhibitor of DHS, with a potential to suppress replication of human immunodeficiency virus type 1 (HIV-1), which exploits activated eIF5A during the formation of progeny viruses (Sommer et al, 2004; Hauber et al, 2005). Recently, the guanylhydrazone CNI-1493 is considered being a promising drug for anti-malarial therapy, because of its dual combined action of inhibition host cell pro-inflammatory cytokine release and parasitic deoxyhypusine synthase (Specht et al, 2008).

Recombinant plant DHS, like its mammalian counterpart, has been shown capable of catalyzing the formation of deoxyhypusine in precursor plant eIF5A (Wang et al, 2001). In Arabidopsis, there is only one DHS gene, but there are multiple members of the eIF5A gene family. It has, therefore, been proposed that the single DHS enzyme activates all of the eIF5A isoforms in Arabidopsis (Duguay et al, 2007), and that DHS function is tightly correlated with the cellular requirement for activated eIF5A (Thompson et al, 2004). DHS expression is likely under tight developmental regulation, presumably by a multi-element promoter that enables its upregulation when there is a need to activate any or all of the eIF5A isoforms (Wang et al, 2003; Duguay et al, 2007). Previous studies have indicated that levels of DHS protein are upregulated during leaf senescence, whether it occurs naturally or is induced prematurely by environmental stress (Wang et al, 2001; Thompson et al, 2004; Wang et al, 2005a). Furthermore, and consistent with the apparent function of DHS, this increase is paralleled by a corresponding increase in one of three isoforms of eIF5A in Arabidopsis, eIF5A-1 (Wang et al, 2003). In a similar vein, levels of DHS and eIF5A protein have been found to increase in tomato leaves in which senescence was induced prematurely by chilling and osmotic stress (Wang et al, 2001). Moreover, constitutive suppression of DHS has been shown to delay the onset of senescence in leaves of Arabidopsis and canola and in tomato fruits (Wang et al, 2003; Wang et al, 2005a; Wang et al, 2005b). However, in each case there were additional pleiotropic effects depending on the degree of suppression. These included enhanced growth, increased tolerance to environmental stress and, in the case of strong suppression, stunted reproductive growth, reduced seed yield and male sterility (Duguay et al, 2007). Moreover, antisense suppression of deoxyhypusine synthase in tomato delays fruit softening, alters growth and development and is involved in the regulation of senescence, a highly regulated process of programmed cell death analogous to apoptosis. (Wang et al, 2005b).

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1.2.3 Deoxyhypusine hydroxylase (DOHH)

The second enzyme, deoxyhypusine hydroxylase (DOHH), hydroxylates the deoxyhypusine residue and eIF5A intermediate to form the hypusine residue and mature eIF5A (Park, 2006). In contrast to the deoxyhypusine-containing protein, no reversal was observed with hypusinecontaining eIF5A, suggesting that hydroxylation at the 4-aminobutyl side chain of the deoxyhypusine residue prevents DHS-mediated reversal of the modification. Whereas the first step of hypusine synthesis is reversible, the second step, DOHH-mediated hydroxylation, locks eIF5A into an active hypusine form, thereby making the overall reaction an irreversible protein modification (Park, 2006) (Figure 3). Although deoxyhypusine hydroxylase activity could be detected in mammalian cells and tissues, the identity of this enzyme remained obscure for many years (Park et al, 2010). The DOHH gene was initially cloned from yeast two-hybrid screening in search of eIF5A binding proteins (Thompson et al, 2003). DOHH exists as a product of a single gene in all eukaryotes and has a unique superhelical structure termed “HEAT-repeat” (Kim et al, 2006b; Park et al, 2010). This enzyme has a nonheme diiron active center that activates O2 (Vu et al, 2009). Since a number of metalchelating inhibitors of DOHH, e.g. mimosine, caused growth inhibition and G1 cell cycle arrest in mammalian cells, DOHH has been assumed to be essential for cell growth (Abbruzzese et al, 1989; Hanauske-Abel et al, 1995). Interestingly, however, the DOHH gene is apparently not essential in the yeast S. cerevisiae, even though endogenous yeast eIF5A mostly exists as the fully modified hypusine form (Park et al, 2006). DOHH seems to be functionally more significant in the fission yeast, Saccharomyces pombe, since a mutation in its DOHH homolog gene caused a temperature-sensitive growth phenotype and altered mitochondrial morphology and distribution (Park et al, 2010). Based on this observation, a role for DOHH, or eIF5A, in micro-tubule assembly and mitochondrial function was implicated. In contrast to yeast, inactivation of DOHH is recessively lethal in multicellular eukaryotes such as C. elegans and D. melanogaster (Spradling et al, 1999; Patel et al, 2009), suggesting a role for DOHH in cell growth and proliferation.

Recently, it was found that the HEAT-like repeats present in the parasite DOHH of malaria differ in number and amino acid identity from its human ortholog and might be of considerable interest for inhibitor design (Kerscher et al, 2010). In plant, DOHH has not yet been described.

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1.2.4 Targeting the hypusination pathway to combat biotic and abiotic stress

Polyamines are required for normal growth of microorganisms including fungi (Foster and Walters, 1993; Havis et al, 1994). Spermidine is known to be the predominant polyamine in fungi. The inhibition of spermidine synthase proved to be effective in reducing fungal growth in culture (Stevens and Winther, 1979). Thus, spermidine analogues were shown to possess antifungal and fungicidal activity in spite of the fact that spermidine inhibitors did not exert their antifungal effects via disruption of other cellular functions associated with polyamines (Jakus et al, 1993; Mackintosh and Walters, 1997a, 1997b, 2003). As the aminobutyl side chain of hypusine is derived from spermidine (Park, 2006; Wolff et al, 2007), spermidine inhibitors inhibit the biosynthesis of hypusine.

eIF5A, DHS, DOHH and their isoforms proved to fulfil a role in the translation of a subset of mRNAs required for specific physiological functions like proliferation, photosynthesis, early development of seedlings, osmotic stress, pathogen attack and senescence induced programmed cell death (Duguay et al, 2007; Hopkins et al, 2008). Characterization of these isoforms will help identifying their role in plant adaptation to stress, in resistance to disease and identifying potential antifungals by disruption of the posttranslational formation of hypusine.

However, five types of resistance to Fusarium head blight have been proposed (Lehoczki-Krsjak et al, 2010): (1) resistance to initial infection, (2) resistance to spread of infection, (3) resistance to kernel infection, (4) tolerance and (5) resistance to toxin accumulation. Only spread in the head and initial infection types of resistance can be used in the search for resistant germplasm and screening breeding lines (El-Badawy, 2001). These practices may enhance adaptation of pathogen population by selecting for aggressive strains and providing a favorable environment which results in disease outbreaks. Prospects to control FHB by chemicals are poor because no fungicides have been found so far to be effective in controlling the disease (El-Badawy, 2001). Resistance breeding is the most economical, environmentally friendly and effective way to control the disease (Jansen et al, 2005). Despite great efforts to find resistance genes against F. graminearum, no completely resistant variety is currently available. Researches are directed towards gaining insight into more details about the pathogen process and reveal spots in the life cycle in order to develop fungicides that can protect host plants from scab infection.

In most crop production regions, high levels of resistance to Fusarium head blight or abiotic stress are not currently available in varieties for planting. However, certain varieties

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have moderate levels of resistance, therefore, breeding for durable resistance against this fungal disease in wheat is the most economical and effective mean of reducing yield mycotoxin contamination causes by this fungus (DeWolf, 2003). The methodology involves the identification of resistance sources and incorporates resistance into genotypes with good agronomic traits. Thus, considerable effort has been devoted to find sources of resistance that can be used in breeding programs (Dunwell, 2005).

Evaluation of barley, wheat, maize and related germplasm yielded only a few accessions with partial resistance to environmental stress (Dahleen et al, 2001). This resistance appears, in most cases, to be polygenic control, making the development of resistant cultivars with suitable agronomic and quality traits a challenge. The insertion of individual antifungal, antitoxin genes and defense genes via genetic transformation has the potential to aid in development of resistant crop cultivars. Among the antifungal genes targeted to combat FHB are coding sequences for proteins that degrade fungal cell walls, disorganize fungal membranes, bolster the host defense response systems, and interfere with fungal protein synthesis, pathogenesis, and/ or accumulation of DON (Dahleen et al, 2001).

Taken together, the biological functions studied so far in eIF5A, DHS, DOHH evaluate highly to characterize these genes in F. graminearum, wheat and maize as hot potential genes and may evidence whether the transgenes achieve their potential against FHB and environmental stress.

1.3 Enhanced methods to study gene expression and function in crops

Different methods have been developed to introduce foreign genes into plants. A common feature is that the transforming DNA has to bypass different membrane barriers; it first has to enter the plant cell by penetrating the plant cell wall and the plasma membrane and must then reach the nucleus and integrate into the resident chromosomes. For the majority of species gene transfer is carried out using plant material competent of regeneration to obtain complete, fertile plants. This implies the development of a tissue culture technology that becomes a special field. Although gene transfer technology has become routine in working with several plant species, in others the limiting step is not the transformation itself, but rather the lack of efficient regeneration protocols (Herrera-Estrella et al, 2005).

The most widely used and successful transformation methods are the nature process of gene transfer by the soil bacterium Agrobacterium tumefaciens and the direct uptake of DNA through particle bombardment.

Cloning, expression and functional characterization of deoxyhypusine synthase from the pathogenic fungus Fusarium graminearum Schwabe (teleomorph Gibberella zeae), wheat (Triticum aestivum L.) and maize (Zea mays L.)